Filoviruses, Ebola (EBOV) and Marburg (MARV), are causative agents of sever hemorrhagic fever in humans with case-fatality rates exceeding 88%. Filoviruses, designated as category A select agents by the Centers for Disease Control and Prevention, are considered potential biowarfare agents and therefore a serious threat to public health and national security. Furthermore, outbreaks in Africa have been on the rise in the past 15 years. There are currently no vaccines or therapeutics available for filovirus hemorrhagic fever. Cellular entry of filoviruses is mediated by the envelope glycoprotein (GP) which is also known to be the primary protective antigen. The main species causing natural outbreaks are Zaire and Sudan EBOV as well as Lake Victoria MARV. A major challenge in development of effective treatment against filoviruses is the extensive sequence diversity within the family. We have recently defined the minimal receptor binding region RBR and demonstrated higher level of homology within this region (>40% between Zaire and Marburg and >80% between Zaire and Sudan). Preliminary data presented in this application demonstrate that an antigen based on the Marburg RBR can protect mice against lethal challenge with Zaire EBOV, the first report of cross protection between these two distant members of filoviruses. Crystallographic studies show that the RBR is masked by the bulky mucin-like domain (MLD). Integrated Biotherapeutics (IBT) has developed engineered antigens for the three major species denoted GPDMuc that expose the RBR by deletion of MLD and demonstrated induction of cross-species antibodies using this antigen. Furthermore, antibody response to this antigen was shown to correlate with protection from lethal challenge in nonhuman primate. In this proposal, we will use these novel antigens along with a powerful and innovative antibody discovery technology (CellSpot), developed by Trellis Bioscience, to identify natural macaque antibodies induced by vaccination against the three filovirus species. CellSpot is a robust and highly miniaturized platform for identification of secreted antibodies from single lymphocytes with the power to select for specificity and affinity against multiple antigens. The technology was successfully used to isolate a rare, cross-strain human antibody to Respiratory Syncytial Virus (RSV) with a 1 pM affinity that is licensed to Medimmune and is now being prepared for human clinical trials. In this project, we propose to use CellSpot to isolate therapeutic antibodies reactive to Zaire, Sudan, and Marburg viruses. The proposal is outlined in three Specific Aims. In Specific Aim 1 we will vaccinate rhesus macaques with filovirus-like particles (VLPs) followed by boosts with purified GPDMuc. Blood will be collected and the serum will be analyzed for a successful cross-species humoral response to GPDMuc (ELISA). In Specific Aim 2 PMBCs of reactive monkeys will be cultivated and analyzed for high-affinity antibodies in the CellSpot platform optimized for antibodies from nonhuman primates. RNA from B cell clones that produce cross-species binding antibodies will be extracted and the heavy and light chains will be cloned into expression vectors for small-scale production in mammalian cells. These candidate antibodies will be screened for antigen binding in a sensitive electrochemiluminescence-based (MSD) and other in vitro assays to identify candidates for testing with live viruses. In Specific Aim 3 we will evaluae the neutralizing activity of the ten best candidates (i.e. highest affinity) in cell culture using ive filoviruses. The 3-5 most promising antibodies will then tested in the lethal challenge mouse models of Ebola and Marburg infection. It is our goal to identify 1-3 antibodies with cross-species neutralizing activity. Upon successful completion of this Phase I SBIR we envision a Phase II focused on demonstration of protective efficacy in NHP models and standard pre-clinical development activities in preparation for regulatory filing.